Assay wells with hydrogel as a well-contents separator

ABSTRACT

In a polymer assay cartridge having wells containing reagents, beads and sample, where the wells are covered (e.g., with Parafilm® or films) and shipped to the point of care, the reagents and well contents can leak out. The reagent solutions are made semi-solid by adding hydrogel reagents and cooling to form a gel. Preferably, the hydrogel is heated before an assay is conducted with the cartridge, and pigmented beads in the wells indicate melting or excessive heating, or congealing of the hydrogel, based on pigment color change.

RELATED APPLICATIONS

This application claims priority to: US Application Serial Nos.: (i)Ser. No. 15/793,490, filed Oct. 25, 2017; (ii) Ser. No. 15/793,445,filed Oct. 25, 2017 (now U.S. Pat. No. 10,126,297); (iii) Ser. No.15/793,384, filed Oct. 25, 2017 (where application (i) claims priorityto Provisional Application Nos. 62/412,745; 62/412,761; and 62/412,751,all filed Oct. 25, 2016; application (ii) claims priority to ProvisionalApplication No. 62/412,734, filed Oct. 25, 2016; application (iii)claims priority to Provisional Application No. 62/412,769, filed Oct.25, 2016)); (iv) Ser. No. 15/390,650, filed Dec. 26, 2016, claimingpriority to Provisional Application No. 62/412,734, filed Oct. 25, 2016,and to Ser. No. 14/992,370, filed Jan. 11, 2016 (now U.S. Pat. No.9,528,985), which is a continuation-in-part of PCT/US2015/013097, filedJan. 27, 2015, claiming priority to Provisional Application 61/932,200,filed Jan. 27, 2014.

GOVERNMENTAL RIGHTS

This invention was made with governmental support under Grant Numbers1R43DA041966-01; and 1R43DA043325-01, both awarded by the NationalInstitute on Drug Abuse. The government has certain rights in theinvention.

BACKGROUND

A reliable, robust assay system which can be deployed to a point of careis useful in a number of settings. For example, where there is aninfectious disease outbreak in a remote area (e.g., the recent Ebolaoutbreaks), such an assay is of benefit for arresting the outbreak asquickly as possible (infected individuals can be quickly located thenisolated) and for keeping health care providers safer.

An assay driver system can be used to automate an assay. One type ofassay driver induces movement of magnetic beads which contact sample (orcontrol) and then carry it into contact with assay reagents. Themovement has to be carefully timed, to ensure proper reaction times indifferent reagents. The results must also be readable for fastinterpretation. One type of discontinuous point of care assay systemwith an assay driver suitable for use herein is disclosed in U.S. Publ'nNo. 2017/0102384 (incorporated by reference). This application disclosesa substantially transparent cartridge having wells containing variousassay reagents. Magnetic beads also reside in the wells, and themagnetic beads are moved among the wells in order to carry sample intocontact with different assay reagents in different wells. It alsodiscloses that a preferred material for sealing one side of thecartridge, in a manner such that only the channels connecting the wellsare open to the outside, and the open side of the wells are sealed, is atranslucent plastic paraffin film, including but not limited toParafilm®. It is noted that the magnetic beads will be attracted towardsthe paraffin film during the assay, and will move along its insidesurface during their movement among the wells. The inner side ofparaffin film allows such movement. The assay is more user-friendly forpoint of care use, if it is shipped to the point of care with themagnetic beads and assay reagents (and preferably, control solution)loaded in the wells—leaving only sample for the point of care operatorto load. The other side of the cartridge, with the openings for thechannels, can be sealed with tape or other material—for shipment orstorage. The tape may or not be needed to be removed to conduct theassay at the point of care.

Even with both sides of the cartridge sealed (by tape on one side and apreferred material on the other), the reagents, magnetic beads or othermaterials in the wells may tend to migrate out of the wells or into thechannels during transport. It is desirable to stop such potentialmigration, so all items start in the appropriate well when the assay isconducted at the point of care. If a gel is used to maintain materialsin wells, it is useful to have a temperature indicator associated withthe assay, so the temperature at which the gel melts—whereupon reagents,magnetic beads or other materials can be moved among wells during theassay—is indicated.

SUMMARY

The migration of reagents among wells and into channels during transportof a loaded assay cartridge can be prevented or inhibited bysubstantially increasing the viscosity of the reagent solutions. Onetype of material which is suitable for such purpose is gelatin hydrogel,which is non-reactive with most reagents. In use, it is mixed with thereagent solutions in the wells.

Preferably, a gelatin hydrogel is selected which is semi-solid at roomtemperature and below (i.e., during shipment) but which is melted toliquefy the reagent solutions at the time of the assay. Theliquification temperature for the gelatin hydrogel should be lower thanthat which affects the reagents or any assay activity, and lower thanthe melting point of the paraffin coating of the Parafilm® (or otherplastic paraffin films or portions of the assay kit).

Some gelatin hydrogels have a melting temperature (i.e., transition fromsolid to liquid) higher than their gelling temperature (i.e., transitionfrom liquid to solid)—which is known as hysteresis. The preferredhydrogels are selected such that the hydrogel-reagent solution has amelting temperature (following gelling) above that encountered duringshipment. Melting of the gelatin (i.e., failure to maintain a gel) couldresult in leakage of materials in the wells during shipment. Duringmanufacturing, the hydrogel-reagent solution is preferably heated tomelting so that reagent solutions can be conveniently placed or injectedinto the wells of the cartridge in liquid form, followed by cooling togel it, before shipment. Among factors affecting the melting/gellingtemperature is the concentration of the hydrogel in the solution—where ahigher concentration is associated with a higher melting and gellingtemperature.

In one embodiment, the hydrogel-reagent solution melts below roomtemperature (RT), so the cartridge can be shipped at refrigerationtemperature (about 4° C.), and then warmed to room temperature justprior to beginning the assay. However, such characteristics aredifficult to achieve. Instead, suitable solutions were made with amelting temperature at about 37° C. and a gelling temperature slightlyabove the range of refrigeration temperatures (around 10° C. to 15° C.).

With these solutions, after heating to melting once and loading theassay wells, the temperature is reduced to about 4° C. to cause gellingof the hydrogel-reagent solution. The cartridge is preferably stored andshipped at 4° C. (which is typically a suitable temperature for storageof assay reagents), and the hydrogel-reagent solution remains gelledduring shipment. Because the melting temperature is about 37° C., thecartridge can tolerate temporary temperature excursions (even up to roomtemperature, or 25° C.) and maintain the gel state. Before the assay isrun, the hydrogel-reagent solution must again be heated to melting—sothe reagents are in solution. The melting temperature is low enough tonot negatively affect reagents or assay performance.

The Bloom number is a measure of the gel strength of gelatin, reflectingthe average molecular weight of its constituents. Gels in commerce canrange up to 300 Bloom. Gels in foods usually range from 125 to 250Bloom. Preferred gelatin hydrogels for use in the invention include highBloom at 1% concentration, as such higher Bloom numbers were found toprovide better control over melting and gelling points.

The Parafilm® (or other plastic paraffin films used as the sealingmaterial) can be adhered to the polymer cartridge, which is normallymolded with a smooth finish in the regions between wells, by rougheningthe normally-smooth polished portions where the sealing materialcontacts, or by using a mold which produces a cartridge with suchroughened regions. More preferably, these portions of the surface haveSPI B-1 grade finish, or approximately a 600 grit finish.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an assay cartridge from the side thatthe wells open to.

FIG. 2 is a plan view of the assay cartridge of FIG. 1.

FIG. 3A is a plan view of the assay cartridge of FIGS. 1 and 2.

FIG. 3B is a sectional view of the assay cartridge of FIG. 3A takenalong the lines A-A′.

FIG. 3C is a sectional view of the assay cartridge of FIG. 3A takenalong the lines B-B′.

FIG. 3D is a sectional view of the assay cartridge of FIG. 3A takenalong the lines C-C′.

FIG. 3E is a sectional view of the assay cartridge of FIG. 3A takenalong the lines D-D′.

FIG. 4A is a plan view of a magnet holder.

FIG. 4B is a cross-sectional view of one embodiment of the magnet holderof FIG. 4A, taken along line C-C′.

FIG. 4C is a cross-sectional view of another embodiment of the magnetholder of FIG. 4A, taken along line C-C′.

FIG. 5A is a perspective view of an assay cartridge assembly with afirst layer and a protective cover layer in place.

FIG. 5B illustrates an exploded view of a first layer and a cover layer,which are both suitable for covering the assay cartridge shown in FIGS.1 to 3E (though a related embodiment of the assay cartridge is shown inFIG. 5B).

FIG. 5C illustrates a perspective view of the assay cartridge assemblyshown in FIG. 5B with the first layer in place and the cover separated.

FIG. 5D illustrates a plan view of one side of the assay cartridge shownin FIG. 5B with the cover in place.

FIG. 5E illustrates the view of the side of the assay cartridge shown inFIG. 5D, where the cover layers are made partially transparent forillustration.

FIG. 5F illustrates a plan view of the inner side of the cover layer.

FIG. 6 is a flow diagram of the steps involved in initiating andperforming an assay with the assay driving apparatus and the assaycartridge shown and described herein.

FIG. 7 is a flow diagram of the instructions for initiating andperforming an assay with the assay driving apparatus and the assaycartridge shown and described herein.

It should be understood that the drawings and the associateddescriptions below are intended only to illustrate one or moreembodiments of the present invention, and not to limit the scope of theinvention. The drawings are not necessarily to scale.

DETAILED DESCRIPTION Definitions

The term “magnetic beads” refers to bead-shaped objects of any size(including microbeads) and composition which can be attracted orrepulsed by a magnetic force, including objects containing paramagneticmaterials or magnetizable materials, such as conductors, and includingconductive metals.

“Plastic paraffin film” includes but is not limited to Parafilm® and anyother product having the recited features.

“Grit finish” and “600 grit finish” include SPI B-1 grade finish and anyother finishes having the recited features.

Referring to FIGS. 1 to 3E, a preferred embodiment of an assay cartridge10, is shown. Assay cartridge 10 has two rows of a series of wells, withthe first well in each row labeled as 12 and 13 respectively, and theremaining wells in one row designated 14, and the remaining wells in theother row designated 15. Each well (12, 13, 14, 15) also has a mini-hole16. Assay cartridge 10 also has a series of channels 18, which extendcompletely through the cartridge 10, and separate each well (12, 13, 14,15) from the well next to it. In this preferred embodiment, there is adepressed section 9 of the cartridge, a hole 7 in corner, and ridges 19around the periphery of a substantial portion of each of the wells (12,13, 14, 15). Ridges 19 function to direct capillary flow around each ofthe ridges, and to prevent fluid from readily migrating between wells.

FIGS. 5A to 5C, and 5E, show a transparent or translucent layer 20(preferably a plastic paraffin film, including but not limited toParafilm® and similar products, which are transparent or translucent andcan adhere to cartridge 10) covers and seals the wells of the relatedcartridge embodiment 11 (which does not include hole 7, depressedsection 9 or ridges 19). Layer 20 is also designed to adhere to theportions of cartridge 10 outside section 9 and to the upper surfaces ofridges 19, to seal the contents of the wells 12, 13, 14, 15 and one sideof the channels 18 (and hole 7) from the surroundings. With translucentlayer 20 in place, channels 18 form air gaps between adjacent wells 12,13, 14, 15 of cartridge 10 (or cartridge 11).

The Parafilm® (or other plastic paraffin films) can be adhered to theappropriate portions of polymer cartridge 10 (or 11) by roughening thenormally-smooth polished portion of the cartridges' surface where thefilm contacts. More preferably, these portions of the surface haveapproximately 600 grit finish, and suitable finishes include SPI B-1grade finish. Alternatively or with such a finish, one could assist inadhering the film to the cartridge using heat and/or pressure, includingpreferably by using a roller laminator to apply pressure and/or heat.

Alternatively, one could do away with Parafilm® and instead use a liquidadhesive on a backing. The liquid adhesive is then spread over thebacking e.g., by heating to reduce the viscosity. The backing can be theimprinted with a readable code 5 (see below), and then spread, e.g., byheating and blowing air, across the backing. To adhere the backing tothe cartridge 10 or 11, the adhesive on the backing is contacted withthe cartridge 10 or 11.

Cover 22 is designed to protect layer 20, especially during transport.Cover 22 is preferably paper or polymer or other material which can beimprinted with a carries a QR code, other readable code, or a barcode 5,and which provides a consistent background color for the last wells (14,15) wherein a color change takes place (see below), and, in some cases,it may be removed from layer 20 before cartridge 10 or 11 is used in anassay. Readable code 5 is preferably printed on the inner side of cover22, so that it can be seen or scanned from at least one side when cover22 is in place on cartridge 10 or 11.

In one example of running an ELISA assay with cartridge 10, samplesolution is introduced into well 12, and a control solution isintroduced into well 15. Preferably, the sample and control are loadedthrough the mini-holes 16 in the respective wells. Magnetic microbeadscoated with antibody against antigens in the control (and which maytarget antigens in the sample, if the sample is positive) are placedinto mini-holes 16 in the wells 14 and 15 which are nearest to wells 12and 13.

Reagents for other steps in the assay (e.g., solutions of labeleddetection antibodies which target and bind to the antigens; solutions todevelop the labels on the detection antibodies into discernable colors)are placed into the mini-holes 16 of other wells 14 and 15. Thesereagents are placed in wells 14 and 15 in a series such that therequisite assay steps are performed as the magnetic beads are moved fromthe wells they first reside in, then to wells 12 and 13 (where somecontact sample and some contact control) and then through the series ofwells 14 and 15.

The wells (12, 13, 14, 15) are then sealed with layer 20, and layer 20is preferably covered by cover 22. The combined thickness of layer 20and the cover 22 is preferably about 100 to 500 microns—though thecombined thickness can vary, with the controlling parameter being theneed to keep the magnets (see below) which act on magnetic beads in thewells during an assay, a specified distance from the magnetic beads.

Prior to using cartridge 10 in an assay, one scans code 5 and sends theinformation to a server/website, which identifies the assay and providesthe instructions about the assay steps and their timing (preferably overthe internet) to a microcomputer which controls movement of magnetbolder 138 (FIGS. 6 and 7). Referring to FIGS. 4A to 4C, magnetic holder138 is preferably a polymer which carries spherical magnets 218 and,optionally, in the embodiment in FIG. 4B, orienting magnets 220, whichalign the polarity of spherical magnets 218. Alternatively, theinstructions can be retrieved from the server/website and manually inputor electronically fed into the microcomputer.

A variety of shapes, sizes and materials can be used to construct thecartridge 10 or 11 itself. Considerations for the materials used in suchcartridges include that it should be substantially transparent, so as toallow the inner side of the cover 22 (with code 5) to be viewed from oneside, and so that the inner side of cover 22 (preferably white oranother suitable background color) can be imaged by scanning.Alternatively, cartridge 10 or 11 itself could be colored for assaycontrast and encoded with code 5, or layer 20 could be colored orotherwise opaque to provide a background for imaging.

Suitable materials for cartridge 10 or 11 include polystyrene,polytetrafluoroethylene (“Teflon®”) and polyethylene. Reagent solutionsin different portions of the capillary are kept separated anddiscontinuous by air gaps 18.

Wells 12, 13, 14, 15 are covered with layer 20, and then it's covered bycover 22, as described above.

Heating and Melting of Hydrogel

The reagents and magnetic beads may then be preloaded in the wells 12,13, 14, 15, preferably being placed (by pipette or capillary action)into the mini-holes 16 in each of wells 12, 13, 14, 15. The reagents andmagnetic beads are preferably in a solution including a gelatinhydrogel, which assists in sealing the contents of wells 12, 13, 14, 15from the open channels 18. Wells 12, 13, 14, 15 are preferably coveredwith layer 20, and then with cover 22, as described above.

In a preferred procedure, a solution including the gel reagents, capableof forming a hydrogel, is first preferably heated sufficiently todissolve any gel which forms. The solution is then cooled sufficiently(e.g., 37° C.) where one can add beads, antibodies or other reagents,without significant risk of denaturation or damage. The solution is thenadded to the wells in cartridge 10 or 11, along with magnetic beads andother assay reagents in appropriate well(s) 12, 13, 14, 15, and thenpreferably cooled to congealing, e.g., about 4° C. The loaded and sealedcartridge 108 (FIG. 5A) can be stored and shipped, preferably with thetemperature held at 4° C. during shipping, or, provided the temperaturein shipping remains below the hydrogel solution melting point, it can beshipped at a higher temperature (e.g., up to RT). As long as thehydrogel remains congealed, the reagent solutions and magnetic beadswill be held in the wells in which they were placed, and will not tendto migrate into other wells.

Before the assay is used, the sealed cartridge 108 is heated to 37° C.to melt the hydrogel solution. The assay can be run at 37° C., or itmight be cooled to RT—provided the hydrogel remains liquid and does notcongeal.

In no case should sealed cartridge 10 be heated as high as the meltingtemperature of the paraffin in layer 20—otherwise, layer 20 will meltinto the wells.

One preferred method to monitor the temperature is by placingcolor-changing thermachromic pigments (available fromSolarColorDust.com) in the wells containing solution; and a morepreferred method is to incorporate or coat the pigments onto beads,which are placed in the wells. The pigment can be incorporated into orcoated onto non-magnetic beads or microbeads, or can be incorporatedinto or coated onto magnetic beads (a core-shell bead with a magneticcore and a pigment-dyed polymer shell is one embodiment); collectively,these are referred to hereinafter as “Pigmented Beads”. In the lattercase, these magnetic beads are different from the magnetic beads used tocarry the ELISA reagent such as a capture antibody.

Examples of using a cartridge of the invention for an assay are below.

Example 1: Using the Cartridge to Perform an Assay

A ready-to-use assay kit is prepared by sealing cartridge 10 or 11 withlayer 20 and cover 22 (forming sealed cartridge 108), and pre-filling itwith reagents and magnetic beads, and including hydrogel and PigmentedBeads, as described above. The cartridge 108 is aligned so cover 22 isdown and so that the magnetic beads rest on the inner surface of layer20.

The degree of heating needed to melt the hydrogel can be controlled, toprevent reagents and coverings on cartridge 108 from exceeding alimit—that limit preferably being well below the temperature at whichassay performance may be affected. During heating, therefore, wherePigmented Beads are in use, one monitors the Beads' color, by viewingthrough a microscope, or using a scanner. Heating is stopped when acolor change indicating melting is observed. Alternatively, one caninclude an additional pigment or pigments in the Pigmented Beads whichchange color to indicate that assay performance may be affected, assayreagents may be affected, or components of the cartridge 108 may melt,if the temperature continues to rise.

One system to perform stopping heating, is to make color-change thecontrol element for a thermostat—such that after heating commencescartridge 108 is observed with a scanner, which, when it detects a colorchange indicating a target temperature, sends a signal to amicroprocessor, whereby power to the heater is cut. At that point, thetemperature of the solution is such that it is liquefied and the assaycan commence, as described below.

In an alternative embodiment, a constant power level is provided to theheating element during the assay such that at equilibrium, the cartridgetemperature is within the range suitable for conducting the assay. Inthis case, once color change is observed by a scanner to indicatereagents have reached a temperature within the desired range, themicroprocessor instructs the device to begin the assay sequence.

Alternatively, the heating of the hydrogel can be carried out in aseparate vessel during the sealing of cartridge 108, and the temperaturemonitored with Pigmented Beads as described above. Once at a suitabletemperature, the hydrogel is added to the wells.

If at any point during the assay, the temperature falls below a limit(as preferably indicated by a third color change in the Pigmented Beads)the scanner can again send a signal to a microprocessor, this time tocause heating of cartridge 108. It is noted that one generally shouldnot excessively heat cartridge 108, but some heat may be provided tomaintain the temperature at an appropriate level below the excessivethreshold.

The magnetic beads are induced to travel from wells 12, 13 to otherwells 14, 15 through channels 18 by moving the holder 138 (and magnets218) in the direction of travel—magnetic beads are drawn towards themagnets 218. The magnetic beads can also be moved so as to mix magneticbeads and reagents by, for example, moving the magnets 218 in adifferent direction from the direction of travel, e.g., so as to inducethe magnetic beads to move back and forth inside one or more of thewells 12, 13, 14, 15.

The movement of the magnetic beads in preferred embodiments is designedto prevent clumping of the magnetic beads and instead move the beadswhile maintaining a bead distribution approaching or achieving a singlelayer over the inner side of layer 20. In one embodiment, mixing of thebeads with the contents of a well is by movement of the beadstransversely to the direction of travel in an oscillating manner. Themixing can also be by oscillating holder 138 in any direction, whetherthe same or different from the direction of travel.

Example 2: Using the Cartridge to Perform an ELISA Immunoassay

After cartridge 10 or 11 is loaded with magnetic antibody-coated beads(preferably, in the wells 14 and 15 nearest wells 12, 13, as notedabove) and reagents suitable for an ELISA are loaded in other wells 14,15, sample is added to well 12, and a control is added to well 13. Insome cases, the control may be pre-loaded along with the magnetic beadsand ELISA reagents. The instructions are executed to induce assay driverto move holder 138 such that the antibody-coated magnetic beads aremoved through channels 18, first to wells 12, 13, and then through wells14, 15, in sequence. The antibody coating on the beads binds to reactiveantigens in the sample or control which reactive antigens are thencarried by the beads.

Oscillating movement of holder 138 in a direction the same, different ortransverse to the direction of travel can be carried out in certainwells, or in all wells in the series, to mix their contents.

It is preferred the strength of the magnetic field acting on magneticbeads and of the acceleration and deceleration of holder 138 is adjustedso that the beads spread out somewhat (like a comet tail) whenoscillating holder 138 to mix beads with the well contents. Optimizationof the movement parameters of holder 138 to achieve rapid mixing ofbeads and well contents is therefore preferred.

Some wells 14, 15, in the series are loaded with detection antibodiescarrying enzyme, or preferably, also with secondary antibodies carryingenzyme. Ultimately, the beads are moved to those wells 14, 15 containingthe substrate for the enzyme carried by the detection or secondaryantibodies, which induces a detectable color change. The color changecan be accomplished with an enzyme substrate combination. In such case,the wells where there is a color change may contain one of thefollowing: PNPP (p-Nitrophenyl Phosphate), ABTS (2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), OPD(o-phenylenediamine dihydrochloride), or TMB(3,3′,5,5′-tetramethylbenzidine)]. One suitable enzyme-substratecombination is horseradish peroxidase (HRP) as the enzyme and TMB as thedetection substrate. When using chemiluminescent chemicals and HRP,which is another option, light is generated as well as a color change.

Some wells 14, 15 may optionally serve as wash chambers to removecontaminants attached non-specifically to the beads—althoughcontaminants on the beads are also removed by passage through the airgaps in channels 18. Oscillating movement of holder 138 in otherdirections can enhance washing the contents of wells which contain washreagents—when the beads reside in those wells.

The concentration of analyte in sample or control solution isproportional to the amount of the analyte that gets attached to theantibody-coated magnetic particles, which in turn is proportional to thenumber of detection antibody molecules that get attached to the analyte.Because the detection or secondary antibodies are attached to HRP, thequantity of detection or secondary antibodies bound to HRP governs therate of catalytic conversion of TMB.

The color concentration can be quantified with a light source anddetector, for example. The final wells can be scanned and either imagedor otherwise quantified for color change, and the representative datacan be transmitted for remote analysis of the assay results, or to thepatient or a designated recipient, as described further below. Asuitable scanner for generating the assay well images is a four channelphotoelectric color sensor, capable of sensing the total light signaland up to three color-filtered signals.

Example 3: Remote Assay Authorization, Performance and Reporting

The assay cartridge 10 or 11 and the system described herein is for usewith an automated assay driver, which actuates and controls performanceof the assay in a secure remotely authorized system. After the assaycartridge 10 or 11 is authenticated (through reading the code 5 andtransmitting it to a secure server or website), instructions aretransmitted to the assay driver, which then controls movement of holder138 in designated directions at designated times, to perform the assay.After the assay is completed, representations of the assay results (fromthe wells where color change took place) are sent from the site of theassay performance for results analysis through the secure system, and/orto a health care worker, and/or to the subject of the assay and/or tohis/her designees (including distribution to anyone who can receive thematerial under applicable HIPAA regulations). The flow of steps in theauthentication and assay process is depicted in FIGS. 6 and 7.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “including”, containing”, etc. are to beread expansively and without limitation. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. It is also noted thatas used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference, and the plural include singularforms, unless the context clearly dictates otherwise. Under nocircumstances may the patent be interpreted to be limited to thespecific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. The terms and expressionsthat have been employed are used as terms of description and not oflimitation, and there is no intent in the use of such terms andexpressions to exclude any equivalent of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention as claimed.Thus, it will be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

What is claimed is:
 1. An assay device stable for shipment comprising: aplurality of wells which are a series of openings connected by firstchannels in a first surface of a body of the assay device, wherein saidwells are filled with assay reagents in a hydrogel solution and at leastone well contains microbeads; at least one air gap in the first channelsto separate fluids in the wells, wherein when gelled, the hydrogelinterfaces with air in the air gap and prevents movement of all assayreagents and the microbeads from the wells into the air gap.
 2. Thedevice of claim 1 wherein the hydrogel is gelatin.
 3. The device ofclaim 2 wherein the gelatin in the solution is at least 150 Bloom. 4.The device of claim 3 wherein the gelatin portion of the solution isless than 10% gelatin.
 5. The device of claim 3 wherein the gelatin inthe solution is Bloom range 180-200.
 6. The device of claim 4 whereinthe gelatin in the solution is at a concentration range of 1 to 4%. 7.The device of claim 1 wherein the microbeads are magnetic microbeads. 8.The device of claim 1 wherein second channels extending transverse tothe first channels intersect the first channels.
 9. The device of claim8 wherein the second channels extend from the first surface through thebody to its opposite surface.
 10. The device of claim 1 wherein plasticparaffin film substantially covers the first surface.
 11. The device ofclaim 1 further including encoding the body with a QR or other code. 12.The device of claim 10 further including adhering a paper or polymerlayer substantially covering said plastic paraffin film.
 13. The deviceof claim 12 further including a QR or other code imprinted on the innerside of the paper or polymer layer.
 14. The device of claim 12 whereinthe plastic paraffin film and the paper or polymer layer combinedthickness is about 100 to 500 microns.
 15. The device of claim 10wherein the plastic paraffin film is adhered to substantially flatportions of said first surface which have an uneven finish.
 16. Thedevice of claim 15 wherein said substantially flat portions have a 600grit finish.
 17. The device of claim 15 wherein said substantially flatportions have a SPI B-1 grade finish.
 18. The device of claim 1 whereinthe body of the assay device is formed of polystyrene,polytetrafluoroethylene (“Teflon®”) or polyethylene.
 19. The device ofclaim 1 wherein the microbeads are magnetic or paramagnetic.
 20. Thedevice of claim 19 wherein the assay device is placed within range ofmagnets which attract the microbeads.
 21. The device of claim 20 whereinthe assay magnets are housed in a moveable magnet holder.